Protein product and process



Patented Dec. 4, 1945 PROTEINPRODUCT AND PROCESS Edwin J. Cohn,Cambridge, Mass., assignor to Research Corporation, New York, N. Y.,

poration of New York No Drawing. Application February 9, 1942, SerialNo. 430,075

22 Claims.

This invention relates to the fractionation of proteins and has for itsobject the provision of improvements in methods for this purpose and ofnovel protein products.

A variety of highly useful proteins is contained, for example, in blood.Some of these proteins are found in the red blood cells, others insolution in the plasma or serum. My invention is directed to theseparation of proteins from blood or other fluids or animal or vegetableextracts; such as milk, liver extract, corn extract, etc.

In my co-pending application Ser. No. 371,401, filed December 23, 1940,of which the present application is a continuation in part, I havedisclosed and claimed processes for fractionating proteins, as well asnovel protein products. These processes involve control of certainfactors aifecting the solubility of proteins and variation of one ormore of these factors. The separation of the proteins is carried out ina liquid medium and involves gradual diffusion through a semipermeablemembrane of a precipitant or precipitants, such as alcohol, at lowtemperatures, into a liquid solution of the proteins to be separated.The temperature, the hydrogen ion concentration, the ionic strength, thenature and the concentration of the precipitant are all carefullycontrolled.

Successive protein fractions may be precipitated, for example frombovine plasma, by the addition thereto of varying amounts of neutralsalts such as phosphates and sulfates, by organic molecules such asethanol, or some other precipitant such as methanol, butanol, acetone, asuitable member of the glycol series, dioxane, etc., or a mixture ofprecipitants, such as alcohols and salts or alcohols and ethers. Furtherfractions may be obtained by variation in the temperature, the hydrogenion concentration and/or the concentration and nature of the salt whichis present.

The plasma may be obtained by first separating the corpuscles from theblood by centrifugation, the flbrinogen being prevented from clotting bythe addition of citratesor like agents.

The protein fractions which are precipitated may, for example, compriselargely fibrinogen, or globulin, or albumin, or mixtures thereof, de-.

1 pending on the selection of the combination of factors afiectingsolubility of the protein.

The fibrinogen separates from the plasma readih when the alcohol orother precipitant is added to it through a capillary or when the plasmais sprayed into the alcohol, precautions being taken to achieve thoroughand instantaa corneous mixing under (conditions such that denaturationof the protein shall be at a minimum. The fibrinogen derived from humanplasma will clot when the other components to blood coagulation areadded to it and can thus be used as a therapeutic agent. Fibrinogen alsoforms plastics which are thermosetting, the properties of which aredescribed in more detail in a co-pending application of John D. Ferry,Ser. No. 430,077, filed February 9, 1942.

The globulins are more labile than the fibrinogen or the albumins, andcertain of the prop- 'erties of some oi these, such as those in normalpooled sera or in convalescent sera, will not in general be retainedunless the greatest precautions are taken in their purification. Thus,although one of the globulins. prothrombin, a factor in the coagulationprocess referred to above, appears to be stable only when preci itatedwith particular precautions, other globulin components such as those ofcomplement are even more labile.

The albumin proteins are of particular interest, as in the treatment ofshock in human beings by in ravenous iniection of solutions of albuminobtained from the blood of man or o1 domestic animals such as the cow,sheen, horse. pig, etc.

Serum albumin is isoelectric near pH 4.8. This is true at least for thealbumin of human, horse and bovine serum or plasma. Since the pH of theblood is maintained near 7.4. the albumins in the blood are far from theisoelectric point, are combined wi h more base per ram than are theglobulins and therefore would have a greater electrophoretic mobilitythan the globulins even were they of the same molecular weight. Themolecular weight of most alubumins is in the nei hborhood of 70.000 orapproximately half that of most of the lobulins. and this also wouldlead to greater electrophoretic mobilities, as well as to greaterosmotic pressures per gram of protein. Although albumins have a greaternet char e per gram at neutral reactions than the globulins, as well asa greater number of charged groups in the isoelectric condition, theseare arranged with far greater symmetry. As a result, albumins haveelectric moments smaller than those of the globulins, and, in fact, ofmost if not all other proteins.

The conditions selected for the fractionation depend upon thesolubilities of the various protein components ofthe system and aredetermined by the five variables: temperature, pH, ionic strength;concentration of precipitant and concentration of the proteincomponents. The

latter factor is of most importance when the concentration of thevarious proteins in the system is high and diminishes in importance indilute protein solutions. These effects of protein concentration oftenprotect labile proteins from denaturation. The other four variables areimportant under all conditions andmust always be under control ifreproducible separations are to be carried out in protein systems. Insufilciently dilute protein solutions they alone suffice to define theseparations. In concentrated solutions the influence of one protein uponanother, depending either upon salt formation or upon the interactionbetween dipolar ions, will have a secondary influence on theseparations. In the case of salt formation, either an increase ordecrease in solubility may result. In the case of interactions betweenproteins as dipolar ions near their isoelectric points, the influence ofone protein upon another depends upon the electric moments of theproteins and will have an additional influence comparable to that of theionic strength of the electrolytes.

The conditions for precipitating successive protein fractions areadvantageously so selected as to alternate the precipitation of thebroadest possible pure fraction with the precipitation of the narrowestpossible impure fraction. Application of this principle gives maximumyields of pure protein products.

The volume fractions of ethanol given are those of the mixtures at 25 C.The pH and ionic strength values in ethanol-water mixtures are taken asthose which would be obtained if the same concentration of salts weredissolved in pure water at 25 C. This convention is used because of theuncertainty involved in any definition of these quantities in theethanol-water mixtures. The pH of the desired protein fractions is takenas that measured with a glass electrode for aqueous solutions of about1% protein concentration.

The separations that I have made are not empirically determined but werechosen with the use of the ultracentrifuge to reveal the size and ofelectrophoresis measurements to record the charge, of the proteins, soas to yield products which should approach homogeneity both with respectto size and net charge, as stated in my aforesaid co-pending applicationSer. No. 371,401.

The fibrinogen is first precipitated from the plasma with ethanol at avolume concentration of 10% as the precipitant, at neutral reaction, thetemperature being below C. and close to the freezing point of thesolution (3 C.)

The gamma globulins, so-called because of their characteristicelectrophoretic mobility, are removed by increasing the ethanolconcentration to5 the temperature being lowered to C.

The alpha and beta globulins are next separated by bringing the pH to5.5-6.0 and the alcohol to 405% by volume, the temperature remaining atC.

The albumin remaining in solution under these conditions is largelyprecipitated from serum or plasma at pH 4.4-4.8 or by lowering thetemperature to 15 C. Albumin, both human and bovine, has been preparedby this method and is pure both electrophoretically and in theultracentrifuge, and there is practically no limit to the amount of thismaterial that can readily be made available.

This method of low temperature alcohol-water fractionation leads tocrystallization of albumin,

where prior methods have not accomplished this. Such crystallization canbe accomplished from plasma which has been contaminated or fromhaemolyzed plasma, thus making it possible to obtain a pure albuminproduct even from contaminated sources.

For certain purposes, it may be convenient to separate a variety ofproteins from the plasma simultaneously. For example, with 25% ethanolat a temperature of 5 C., the pH remaining unadjusted, fibrinogen andgamma globulins are precipitated together. The protein product soobtained may be used in the manufacture of plastic compositions.

The solubility of each protein in human or in animal plasma will beminimal in the neighborhood of its isoelectric point. The separation ofglobulins or of casein from water by the addition of suflicient acid oralkali to bring the protein to its isoelectric point illustrates theimportance of pH.

Many proteins, notably albumins, are soluble in water, even at theirisoelectric points. These may be precipitated either near theirisoelectric points or in a pH region not too far distant from theirisoelectric points, by salt precipitation or by the addition of anorganic precipitant, such as an alcohol, acetone, methanol, butanol,dioxane, a suitable member of the glycol series, etc. The amount of suchprecipitant necessary will in general be minimal at or near theisoelectric point of the protein. Increases in temperature will have theeffect of increasing solubility in the case of proteins uncombined withacid or base near their isoelectric points. Organic solvents will ingeneral have a greater precipitating action on proteins the lower thetemperature. Sufiiciently concentrated neutral salts such as phosphatesand sulfates are often of the reverse effect, the protein solubilitydecreasing with increasing temperature.

In the case of neutral salts part of the precipitating action iscomparable to that of the organic molecule, but part of the salt actiongives rise to an increase in the solubility with increase in theconcentration of ions (ionic strength). This is the influence of theionic strength which must be taken into account also as an importantvariable when organic solvents are used as pro tein precipitants. Thusin the separation of the albumins from the globulins in plasma thecontrol not only of the concentration and of the organic solvent but ofthe temperature, the pH and the ionic strength, is essential for thesatisfactory separation and for the crystallization of' .the albumin.

A convenient method for controlling both pH and ionic strength with thesame reagent is in the use of buffer solutions. Thus phosphate oracetate bufiers of fixed pH and ionic strength have been used for thispurpose under a variety of conditions.

The influence of the four variables noted above may be illustrated asfollows:

(1) If the alcohol concentration of the plasma solution is brought to40% by volume at room temperature, the fibrinogen and gamma globulinswould be precipitated and in part denatured, but the alpha and betaglobulins and the albumin would not be completely precipitated from theplasma.

(2) Decrease in temperature to 5 will very much diminish denaturation,will increase the precipitation of the globulins, but will still notcompletely precipitate the alpha and beta globisoelectric points liebetween p115 pH close to 5.5, the precipitation of large parts of thealpha and beta globulins is achieved.

(4) The extent of this precipitation will still depend, however, uponthe ionic strength, increasein ionic strength increasing the solubilitynot only of the globulins but of the albumin. Th amount of this effectwill diil'er, for the globulins and the albumins, and sincethe'globulins are far less soluble in 40% ethanol throughout this pHrange an ionic strength of 0.05 has been used to increase the solubilityof the albumin sumciently without increasing the solubility of theglobulin to the extent where separation is unsatisfactory.

At this pH, alcohol concentration, ionic strength and temperature,albumin is suillciently soluble to be almost quantitatively extractedfrom the precipitate if these proteins are at approximately one quarterof the concentration that they would have had in the original plasma.Thus the albumin concentration is approximately grams/liter, while theglobulin soluble under the same conditions never exceeds onehalfgram/liter and ii the separation is carefully carried out is oftenlower than 0.10 gram.

.As a further example 01' the influence oi the pH at constant ionicstrength, I may then precipitate albumin at constant ionic strength bybringing the pH close to the isoelectric point of the albumin. namely pH4.7, temperature and alcohol concentration being maintained constant. Inorder not to expose the albumin to even a local excess of acid, Iachieve this precipitation by the addition of an acetate buffer of pH4.2, the ionic strength being changed from 0.05 to 0.06, while the pH ischanged from 5.5 to 4.7.

In adding increasing amounts of alcohol and in varying the temperaturefor the precipitation of successive protein fractions, the temperatureand the percentage of alcohol may be so correlated that the temperatureused is just above the freezing point of the solution at the percentageof alcohol present. I

The fibrinogen fraction. when redissolved, gives a solution whichexhibits double refraction of flow. The fibrinogen consists oi moleculeswhich sediment in the ultracentrifuge in 0.2 molal potassium chloridewith a velocity constant of 7.0 to 7 9X10, migrate in theelectrophoresis apparatus at 0 C. in a phosphate buffer solution ofionic stren th 0.2 and pH 7.7 with a mobility of 1.8 to 2.3X10, arerod-shaped, and thus yield very viscous solutions. This product has theproperty of coagulating to form the clot charaee teristic of the bloodclot in the presence of calcium andprothrombin. Prothrombin is aconstituent of another protein fraction of plasma.

One globulin fraction has t e pro erty of polymerizing to formaggregates'of varying molecu ar weight. This protein generally has asedimentation constant in the u tracentrii'uae of about s=18 10- or s=1210 and its m lecules are also rod-shaped and reveal double refraction offlow.

Although the proteins that separate from 15% ethanol when thetemperature is shifted from 0 to -5 C. and the further precipitate thatsepcreased to 20% or 22% ethanol at 5 C. consist of a number oi'diflerent chemical individuals, some oi! isoelectric point near 7,othersof more acid isoelectric points, some near 6, some euglobulin and somepseudoglobulin, the fraction as a whole appears to be very uniform withrespect to electrophoretic mobility, as judged in phosphate buil'ers ofionic strength 0.2 and pH 7.7, the mobility constant having the value0.8 to

2.2x10-' cm. persecond at 0 C. The size of themolecules in this fractionis also constant with the exception of a small amount of the highmolecular weight component of sedimentation constant 12 or -18 10- thesedimentation constant of the rest of the fraction being about'5.9 to6.7x 10- in 0.2 molal potassium chloride. This fraction solublein saltsolutions but in large part precipltable and separable into its variouscomponents by dialysis at varying pH, has been prepared as a copious,white, odorless precipitate. It is less soluble and more viscous thanthe albumin fractions and of very low electrical net charge.

As contrasted with the protein fractions made up of asymmetric moleculesof high viscosity, which are precipitated by the lower concentrations ofethanol mixed with water in neutral solution, the fraction separated atacid reactions (near pH 5) from 40% ethanol at 5 C. consists almostcompletely of albumin of sedimentation constant in 0.2 molal potassiumchloride of 4.0 to 4.4 l0- and of electrophoretic mobility at 0 C. inphosphate buffer 01' ionic strength 0.2 and DH 7.701 4.7 to 6.0X10- themolecules being far more nearly spherical in shape and far more $01-uble, dissolving readily in water to* form a limpld 20% solution of lowviscosity.

In an alternate procedure, any desired protein fraction or group offractions may be obtained by'extraction from a solid mixture. Thus I maydry any mixture of these proteins, obtained by any of the procedurespreviously mentioned. I may then obtain the albumin fraction, forexample, from this dried plasma by extraction under conditions such thatonly the albumins are soluble, the other proteins being insoluble. Thealbumin can then be separated by treating the albuminsolution in amanner similar to that described previously. The set of equilibriumconditions at which extraction 01' any desired fraction from the solidmixture is accomplished may be selected at will by the choice of properpH, temperature, ionic strength and alcohol concentration. and thesolution thus obtained may be treated for the further subdivision offractions contained therein in the manner previously discussed.

In additioifsuccessive extractions may be made from the/dry mixture ofproteins, with different solvents and under different temperature, ionicand pH conditions.

Some protein precipitants, such as alcohol, have a tendency to denaturemany proteins with wh ch they come in contact, the danger ofdenaturation increasing with concentration of the alcohol and increasein temperature. For many proteins. it has been found advisable toexercise considerable care in mix ng the precipitant withthe plasma orother protein solution in order to avoid denaturation of the protein.For these. the procedures disclosed in my co-pending application Ser.No. 371,401 are to be recommended.

In these procedures the precipitant (e. 2.. ethanol) is added by diffuson through a semi-permeable membrane. While maintaining the plasma atslow temperature, for example at 0 C., I

may suspend in it a sac of Cellophane which contains a solution ofethanol and also other ingredients as herein indicated. Assuming thatthe first protein fraction is to be taken at ethanol,

the amount of ethanol in the solution within the semi-permeable membranewill be' so calculated with regard to the total volume or liquid bothinside and outside the membrane that, when equilibrium is reached, theplasma will contain exactly 10% alcohol.

It is often convenient to precool the alcohol or other reagent, belowthe temperature of the plasma, so that the temperature of the mixture atequilibrium will be close to the freezing point. This is an additionalprecaution against denaturation.

Instead of placing the precipitant within the semi-permeable membraneand suspending it, in theplasma, the plasma may be confined within themembrane and suspended in a bath of the precipitant. In either case,stirring of the plasma is advisable and is usually necessary, and if theplasma is placed within the membrane, stirring of the bath andrecirculation by pumping of the precipitant is also recommended. Thepurpose of the stirring is to prevent undesirably high concentration ofthe precipitant at the interface between the plasma and the precipitant.

Procedures involving the use of a semi-permeable membrane are useful notonly for the addition of precipitants, but also for the addition of anyreagent for modifying or purifyin proteins, if such reagent threatens todenature the protein. For example, acid chlorides or acid anhydrides,which combine with the amino and phenolic hydroxyl groups of proteins,iodine which forms iodo proteins, or pyridine or other bases whichmodify proteins but which tend to cause denaturation, may be added throuh a semi-permeable membrane in order to avoid denaturation of theproteins.

Suitable large-scale equipment, in which Cellophane sheets separate theplasma from the precipitant, will generally be found convenient inproduction on a commercial scale.

Instead of Cellophane, collodion or sausage casings, or the like may beused as the semi-permeable membrane. However, collodion cannot be usedwhen alcohol is the precipitant, since it is soluble in alcohol.

Some proteins, however, will be found to have a much greater resistanceto denaturation by alcohol. Albumins. for example, as indicated above.have this property. When the desired product is a protein of thischaracter, it is possible to use procedures which would not be suitablein the production of other and less stable proteins such as, forexample, many globulins. The precipitant (e. g., ethanol) may in thecase of these more stable proteins, such as albumin and fibrinogen, beadded directly to the albumin solution without the use of asemi-permeable membrane. It is,

in fact, possible to purify albumin of certain other protein impurities,such as certain globulins, by permitt ng the globulins to denature in.

to ethanol at pH 4.8 at temperatures between 0 C. and room temperature,separating the precipitated, denatured protein impurit es and leavingthe purified albumin in solution. This albumin may be precipitated incrystalline form, under the proper conditionsas hereinafter described.

When the stability of the desired protein product permits addition ofthe alcohol without Thus flbrinogen' been precipitated from plasma byspraying the plasma into ethanolwater mixtures, or ,vice versa. Aflowing :Iunction has also been employed for this addition, by supplyingethanol-water mixtures through one end of the top of a T-shaped tube,and plasma through the other end of the top. The two solutions arethoroughly mixed in this tube, and pass together down the stream of theT. Alcohol has. also been added by forcing ethanolwater mixtures, or insome cases pure ethanol, through a tube of small diameter (e. g. ofcapillary size or larger) or a sintered glass plate, into the proteinsolution, with stirring. The precautions'against denaturation necessaryin 8.1'1y0f these additions depend upon the stability of the desiredprotein, product or products. Very labile proteins can be obtained in anundenatured state by careful avoidance of any local excess of alcoholconcentration. The rate of denaturation of proteins by local excess ofalcohol increases greatly with increasing temperature.

The method of addition of the precipitant to the protein, as well as thenature of the protein, will determine the degree of denaturation as wellas the state of subdivision of the precipitate; the latter is of greatimportance in the processing of proteins on a commercial scale.

The stability of the albumin is such that I find it sufiicient to addthe buffer in many of the above processes through a capillary withstirring without resorting to the use of a semipermeable membrane. Sinceprotein precipitates tend to be either very finely divided or sticky,they thus present a problem in obtaining a precipitate which can besatisfactorily collected. Rapid addition of the precipitant, as by theabove or similar methods, or even bypouring it into the albuminsolution, results in the formation of coarser protein precipitates,which are easier to handle.

Precipitated proteins may be dried by freezing the wet precipitate aswith liquid air or solid carbon dioxide, then subjecting the solidfrozen mass to a vacuum, whereby the solvent is removed by vaporizationand the protein is obtained in the form of a dry powder. Due to therelatively much greater stability of albumin, however, it has been foundpossible to dry albumin and other more stable proteins under vacuum atroom temperature or by passing dry gases over I the wet precipitates, orby removing the water by organic solvents using conventional methods.

The albumin precipitate is a colorless white powder, free of reducingsubstances. When sufflciently purified it readily dissolves in water,yielding clear solutions even at concentrations as great as 70% proteinby volume. In fact, it is apparently completely miscible with water. Noprecipitate appears in concentrated solutions even after a considerableperiod of time. Thus 25% solutions have been maintained at 37 C. for,over two months without clouding up and at 45 C. for over a month.Human albumin solutions which have been kept for these lengths of timeunder these conditions have been injected into man as transfusion mediawithout giving any untoward reactions. Albumin purified in this wayappears to be stable for considerable periods of time even in 20%ethanol at room temperature.

Indeed, the albumin is so stable that instead of concentrating it byisoelectric precipitation from 40% ethanol, it may be concentrated bylow temperature vacuum distillation even under 2,390,074 r 5 conditionssuch that the-globulins would be dethe precipitation or extraction orany protein natured- Thisunexpected discovery of the stafraction willdepend upon the protein or probility of the albumin even inethanol-water mixteins desired for the purpose in mind. Those tures attemperatures above has simplified the metho and concentration. I 1

Even bovine serum albumin purified bymy process, which takes advantageof the stability of albumin being greater than that of the globulins,can be crystallized under certain conditions. I may crystallize thealbumin by redissolving the previously precipitated albumin in 40%ethanol at C. and pH 5.5-6.0, using an ionic strength greater than waspreviously employed so that the solubility of the albumin solution shallbe far greater than before. If the ionic strength of the acetate bufieris 0.3, I can obtain a 20% solution of the albumin component in 40%ethanol at pH 5.5 at 0 C. If the temperature is C. and the alcohol ismaintained at 40% under a pH of 5.5-6.0, the ionic strength of theacetate buffer must be higher, in the neighborhood of 0.5. Uponstanding, crystals separate from such solutions. Were the ionic strengthor temperature higher, these would be dissolved. At a lower alcoholconcentration (say 15%) at this temperature and pH, part at least of thecrystals could be recrystallized if the ionic strength were lower than0.01. The carbohydrate content of such crystals will be less than 0.1%.

It should be remembered that when the albumins are separated from theglobulins at pH 5.5-6.0, temperature 5 C. and alcoholic concentration40%, the ionic strength is as low as 0.05 and the albumin is present inthe mother liquor to an extent less than 1%. By increasing the ionicstrength ten-fold, one may produce a far greater increase in thesolubility of the proteins, the logarithm of the solubility rather thanthe solubility being a function of the ionic strength in ethanol-watermixtures. It is from these highly concentrated protein solutions thatcrystallization takes place with the greatest case.

By successively recrystallizing at lower ionic strengths and loweralcoholic concentrations, I may remove impurities which have differentsolubilities under these different conditions.

If the same conditions of temperature, pH and alcohol concentration areemployed, lower ionic strength should be used in crystallizing humanthan bovine albumin. Thus I have found that at 40% ethanol at. 5 and pH5.5-6.0, an ionic strength of 0.2 will suiiice. If the crystals formedunder these conditions be dissolved in a minimal quantity of water and40% ethanol be added to the solution until turbidity is reached,crystals will again form, crystallization this time taking place at alower alcoholic concentration and ionic strength. This process I mayrepeat; each time crystallizing from a lower ionic strength and loweralcoholic concentration.

It is also possible to crystallize from the mother liquors of each ofthese crystalline masses by adjusting the pH to a slightly more acidreaction or by lowering the temperature. This I also sometimes findconvenient, although it is generally more convenient to combine themother liquors, precipitate the albumin at its iso-electric point by themethod previously described, collect the precipitate by filtration orcentrifugation, and dissolve the precipitate at the higher ionicstrength in 40% ethanol. The process of recrystallization must then becarried out as described above.

The particular set of conditions selected for zero degrees C. thus s forits purification conditions given in the ioregoing specification havebeen by way of example, As has been indicated, many other sets ofconditions are possible, by which diiierent protein fractions ma beobtained, in varying degrees of purity.

Having thus described my invention, I claim:

A method of tractionating proteins by precipitation from a solutioncontaining a plurality of protein fractions, said solution having a pHabove the iso-electric point of the fraction desired to be precipitated,which comprises lowering the pH of the solution to bring the same toapproximately the iso-electric point of the desired fraction to beprecipitated, bringing the ionicstrength of the soiutionto between 0.01and 0.2, lowering the temperature of the solution to betweenapproximately 0 C. and the ireezing point or the solution, adding anorganic precipitant for protein to the protein solution, the amount ofthe precipitant added being such as to cause precipitation of thedesired fraction only from the protein solution at the said temperature,and separating the precipitate from the solution.

2. A method of fractionating proteins by precipitation from a solutioncontaining a plurality of protein fractions, which method comprisesbringing the pH of the solution to approximately the iso-electric pointof the desired protein fraction to be precipitated, bringing the ionicstrength of the solution to-between 0.01 and 0.2, lowering thetemperature of the solution to between approximately 0 C. and thefreezing point of the solution, adding an organic precipitant forprotein to the protein solution, the amount of the precipitant added,the pH, the ionic strength and the temperature being such as to causeprecipitation of only the desired fraction from the protein solution,and separating the precipitate from the solution.

3. The method or claim 2 precipitant is alcohol.

4. The method of claim 2 in precipitant is acetone,

5. The method of claim 2 precipitant is dioxane.

6. In a method for fractionating proteins, the steps which comprisemixing with a solution of proteins an organic precipitant for proteinadjusting the temperature between 0 and -15 0., the amount ofprecipitant between 10% and 40%, the pH between 4.4 and 7 and the ionicstrength in which the organic which the organic in which the organicbetween 0.05 and 0.2, and separating from the resulting liquid systemwhich is insoluble therein.

7. In a method for fractionating proteins, the steps which comprisemixing with a solution of proteins an organic precipitant for protein,adjusting and maintaining the temperature of the protein solution abovethe freezing point thereof but not above 0 C., the amount of saidprecipitant between 10% and 40% of the solution, the pH between 4.4 and7, and the ionic strength between 0.05 and 0.2, and separating from theresulting liquid system a protein which is insoluble therein.

8. In a method for fractionating proteins, the steps which compriseadding to a solution containing a mixture of proteins, both anelectrolyte and an organic precipitant for protein, the electrolytebeing added in amount sufficient to bring the ionic strength to between0.01 and 0.2 and a protein precipitate the precip tant being added inamount such as to cause precipitation of only the desired proteinfraction, adjusting and maintaining the pH of said solution between 4.4and '1 and the temperature thereof between and 15 C. and therebyprecipitating a protein from the resulting sy tem.

9. The method of purifying and crystallizing albumin, which comprisesdissolving impure albumin in an alcohol solution containing from 15 to40% alcohol, at a pH of approximately 5.5 to 6.0, an ionic strength of0.05 to 0.5 and at a temperature of 0 C. to 0., and maintaining saidsolution within said temperature range until a purified albumincrystallizes out.

10. As a new product, an aqueous albumin solution containing at least25% by volume of albumin and suitable for intravenous injection, whichsolution remains stable without precipitation of the albumin afterexposure to a temperature of 45 C. for a period of one month.

11. The method of fractionating substances which have differingsolubilities, all of said substances being prevented from passingthrough a semi-permeable membrane, which comprises diifusing an organicprecipitant through a semipermeable membrane into a solution of amixture of said substances, at a controlled temperature and hydrogen ionconcentration, removing the precipitate thus formed and precipitating aplurality of successive fractions of said substances by variation in oneor more of said factors.

12. The method of preventing denaturation of proteins by modifyingreagents which would normally result in denaturation, which comprisesadding the reagents to a protein solution by diffusion through asemi-permeable membrane.

13. The method of crystallizing albumin from a protein solution whichincludes diffusing alcohol through a semi-permeable membrane into suchsolution, under conditions of controlled hydrogen ion concentration,temperature and ionic V strength, to cause separation of albumincrystals therefrom.

14. In a method for fractionating proteins, the steps which comprisediffusing an organic precipitant through a semi-permeable membrane intoa solution of proteins, controlling the amount of said precipitant inthe solution, the temperature, the hydrogen ion concentration and theionic strength, separating the resulting precipitate from the proteinsolution, and separating successive protein fractions by varying aplurality of said factors affecting solubility thereof.

15. In a method for fractionating proteins, the step which comprisesdiffusing alcohol through a semi-permeable membrane into a solution ofproteins, at a. temperature below 0 C.

16. In a method for fractionating proteins, the step which comprisesdiffusing alcohol through a Cellophane membrane into a protein solution,at a temperature not exceeding 0 C.

17. The method of fractionating proteins which comprises diffusing intoa protein solution,

through a Cellophane membrane, a precipitant comprising alcohol and abutter, until equilibrium is reached on both sides of said membrane withregard to the alcohol and the buffer.

18. The method of fractionating proteins from blood plasma to atemperature in the neighborhood of 0 C. or lower, diffusing alcoholthrough a semi-permeable membrane into said plasma, whil maintained atsaid temperature, separating the re sulting protein precipitate,dialyzing additional alcohol into the remaining solution, removing theresulting precipitate, reducing the temperature of the plasmaandseparating subsequent protein fractions by dialyzing further amountsof alcohol into said plasma.

19. The method of fractionating proteins which comprises separating aprotein solution and an organic precipitant therefor by a semi-permeablemembrane which is permeable to the precipitant but not to the protein,diffusing the precipitant through the membrane into the protein solutionunder conditions of controlled hydrogen ion concentration, temperatureand ionic strength, and agitating the protein solution at the interfaceto prevent any local excess of the precipitant.

20. The method of fractionating proteins from blood which comprises assteps removing the corpuscles from the blood b centrifugation whilepreventing clotting of the fibrinogen by the addition of ananti-clotting agent, then precipitating a protein from the remainingplasma by diffusing an organic precipitant through a semipermeablemembrane into the said plasma, and by adjustment of the hydrogen ionconcentration, the temperature and the ionic strength of the plasma,then drying the precipitate so obtained.

21. In a method for fractionating proteins contained in blood, the stepswhich comprise separating the corpuscles from the blood, treating theremaining plasma for the separation therefrom of a series of differentproteins by systematic variation of the hydrogen ion concentration, thetemeprature, the ionic strength and the alcohol content of the plasma,said separation involving diffusion of alcohol into the plasma through asemi-permeable membrane,

22. The method of fractionating proteins from blood plasma whichcomprises as steps precipitating a plurality of different proteinfractions from the plasma by diffusing alcohol into the plasma through asemi-permeable membrane and by varying the temperature of said plasma,the temperature being progressively lowered and the alcoholconcentration of the plasma being increased, with the precipitation ofsuccessive protein fractions, the temperature and the percentage ofalcohol being so correlated that the temperature employed for theprecipitation of any given protein fraction is close to but above thefreezing point of the plasma at the percentage of alcohol presenttherein.

EDWIN J. COHN.

which involves cooling said plasma.

CERTIFICATE OF CORRECTION. 7 Patent No. 2,590,07 December 11., 19LL5.

EDWIN J. COHN.

It is hereby certified that error appears in the printed specificationof the above numbered patent requiring cerrectionas follows: Page 1.1.,second column, line 9, for "stream" read stem-; and page 6, secondcolumn, line L15, claim 21, for "temeprature" read -temperature--; andthat the said Letters Patent should be read with this correction thereinthat the same-may conform to the record of the case in the PatentOffice.

Signed and sealed. this 26th day of February, A, D, 19146.

Leslie Frazer (Seal) First Assistant Commissioner of Patents

